Summary Normal motor activity in the gastrointestinal (GI) tract relies upon coordinated contractions of the smooth muscle cells (SMCs) that line its walls. The ultimate contractile behavior of GI muscles depends upon the state of SMC excitability when regulatory inputs (e.g. nerve reflexes, hormones, etc) are applied. This project has developed the concept of the SIP syncytium, an electrically coupled tissue consisting of SMCs, interstitial cells of Cajal and PDGFRa+ cells, that establishes the basal excitability of smooth muscles, organizes contractile behavior into phasic contractions, and transduces signals from a variety of motor neurons. GI motility in fact results from the integrated output of the SIP syncytium. During the past funding period we discovered that PDGFRa+ cells are a novel class of regulatory cells and essential component of the SIP syncytium. These cells express receptors and ion channels consistent with mediation of purinergic inhibitory regulation in the GI tract. Through isolation and purification of PDGFRa+ cells by FACS we performed deep sequencing to better understand the phenotypes of these cells. The expression data suggest that many additional pathways, such as adrenergic and inhibitory neuropeptides, contribute to the regulatory functions of PDGFRa+ cells. The focus of the upcoming funding period is to test the hypothesis that sympathetic nerve and enteric inhibitory neuropeptide signals are also transduced by PDGFRa+ cells, making these cells a convergent central integrator of inhibitory regulation of colonic motility. We will first clarify the tissue level responses mediated by sympathetic neurotransmission and inhibitory neuropeptides that can be attributed to mechanisms expressed uniquely by PDGFRa+ cells. Preliminary data clearly show direct sympathetic innervation and inhibitory effects of PDGFRa+ cells powerful enough to halt propulsive contractions in the colon. This is an exciting new concept of how stressful conditions might radically alter bowel habits through PDGFRa+ cells. Our findings suggest that any stimulus coupled to Ca2+ transients in PDGFRa+ cells will activate small conductance Ca2+-activated K+ channels and initiate powerful hyperpolarization and inhibitory influences on the SIP syncytium. We will use cutting edge Ca2+ imaging techniques, including cell-specific optogenetic sensors, to better understand the cells and sequence of events that occurs in peptidergic and sympathetic inhibitory responses mediated by PDGFRa+ cells. Finally, in studies of single cells we will utilize electrophysiological and imaging techniques to expand our knowledge of ionic conductances that might be used by PDGFRa+ cells to generate integrated inhibitory regulation. We will also study the role of Ca2+ stores in the responses and how stores are maintained for long term and repetitive inhibitory responses. Completion of the specific aims will provide novel information about an important cell type never been considered a regulatory factor in GI motility. Knowledge of the regulatory mechanisms contributed by PDGFRa+ cells will provide new understanding about factors that generate normal colonic possibly discover new methods for therapeutic management of motility disorders. OMB No. 0925-0001/0002 (Rev. 03/16 Approved Through 10/31/2018) Page Continuation Format Page